Mechanistical Insights into the Bioconjugation Reaction of Triazolinediones with Tyrosine

Abstract

The bioconjugation at tyrosine residues using cyclic diazodicarboxamides, especially 4-substituted 3 H-1,2,4-triazole-3,5(4 H)-dione (PTAD), is a highly enabling synthetic reaction because it can be employed for orthogonal and site-selective (multi)functionalizations of native peptides and proteins. Despite its importance, the underlying mechanisms have not been thoroughly investigated. The reaction can proceed along four distinctive pathways: (i) the SAr path, (ii) along a pericyclic group transfer pathway (a classical ene reaction), (iii) along a stepwise reaction path, or (iv) along an unusual higher order concerted pericyclic mechanism. The product mixtures obtained from reactions of PTAD with 2,4-unsubstituted phenolate support the SAr mechanism, but it remains unclear if other mechanisms also take place. In the present work, the various mechanisms are compared using high-level quantum chemistry approaches for the model reaction of 4 H,3 H-1,2,4-triazole-3,5(4 H)-dione (HTAD) with p-cresol and p-cresolate. In a protic solvent (water), the barriers of the SAr mechanism and the ene reaction are similar but still too high to explain the available experimental observations. This is only possible if the SAr reaction of cresolate with HTAD is taken into account for which nearly vanishing barriers are computed. This satisfactorily explains measured conversion rates in buffered aqueous solutions and the strong activation effects observed upon addition of bases.

Tyrosine Conjugation Methods for Protein Labelling

“…Although TAD grafting to Tyr is generally described in the literature as an ene‐like reaction, an S E Ar mechanism most likely takes place (Figure 19). Indeed, the reaction shows higher conversion yields at basic pH, [82] probably because of higher phenolate reactivity in an S E Ar mechanism, as predicted by a recently published quantum chemical approach [83] …”

“…Indeed, the reaction shows higher conversion yields at basic pH, [82] probably because of higher phenolate reactivity in an S E Ar mechanism, as predicted by a recently published quantum chemical approach. [83] The versatility of cyclic diazodicarboxamides for Tyr bioconjugation was first reported ten years ago by the Barbas' research group at the Scripps research institute. [82] Since then, published examples of successful phenyltriazolinedione (PTAD) conjugations have flourished, not only in the field of chemical biology but also in materials chemistry.…”

Discriminating changes in protein structure using PTAD conjugation to tyrosine

“…We assessed the products of PTAD conjugation with tyrosine to determine products our experiment designs may produce. Previous reports noted such additional reactions as conjugates with amines, second additions to tyrosine phenols, and short-lived conjugations to cysteine residues 15,16 . We confirmed by 1 H NMR the reaction with a tyrosine analogue, 3-(4-hydroxyphenyl)propionic acid (Supplemental Spectra 1).…”

Bio-orthogonal triazolinedione (TAD) crosslinked protein nanocapsules affect protein adsorption and cell interaction

“…8,13 Herein we report the synthesis of protein nanocapsules that were crosslinked with a 4-phenyl-triazolinedione (TAD) derivative, which specifically reacts with amino acids with delocalized π-electron systems. [14][15][16][17] The ability of TAD to act as a strong dienophile in a Diels-Alder reaction has already been shown almost 60 years ago. 18 Recent studies performed by Du Prez and coworkers again showed its versatility in current applications in polymer science.…”